The Ca2+ translocating ATPase (i.e., the Ca2+ pump) is an integral membrane enzyme that transports Ca2+ ions into the sarcoplasmic reticulum (SR) to promote muscle relaxation. In cardiac muscle, the Ca2+ pump is regulated by phospholamban (PLB), and this regulation is relieved by PLB phosphorylation. During normal aging, Ca2+ pump activity declines resulting in prolonged Ca2+ transients and slowed cardiac muscle relaxation rates. In addition to other well defined age-based changes in Ca2+ pump function, the Ca2+ pump is progressively nitrated at specific tyrosine residues during aging, and studies of the isolated enzyme suggest tyrosine nitration correlates directly with Ca2+ transport inhibition. However, the mechanism of Ca2+ pump inhibition following nitration is not understood, so a role for calcium pump nitration in age-based loss of calcium pump function cannot be supported or refuted. Therefore, the objective of this application is to determine the physical and kinetic mechanisms by which Ca2+ pump nitration leads to the inhibition of Ca2+ pump transport activity. Our hypothesis is that Ca2+ pump nitration shifts the enzyme toward the low energy, Ca2+ affinity Ca2+ pump state (the E2 intermediate) relative to the high affinity, Ca2+-activated Ca2+ pump state (the E1 intermediate), and inhibits the kinetic transitions between the two enzyme intermediate states. Because PLB interacts most strongly with the E2 state, we also propose that PLB will be a more potent inhibitor of the nitrated Ca2+ pump. To test these hypotheses and their alternatives, we will combine kinetic studies of the Ca2+ transport cycle (Aim 1) and spectroscopic studies of Ca2+ pump conformational changes during the Ca2+ transport cycle (Aim 2) to determine the physical mechanism of Ca2+ pump inhibition due to nitration. For these studies, we will use native cardiac SR vesicles obtained from young adult (6 month old) rats versus senescent (24 month old) rats, which contain different amounts of endogenously nitrated Ca2+ pump. The specific role of Ca2+ pump nitration in physical and kinetic differences between the two sample types will be further explored by conducting additional studies on Ca2+ pump from the young animals pre-treated with peroxynitrite to mimic progressive age-based Ca2+ pump nitration. The results of this project will define or reject a role for Ca2+ pump nitrotyrosine formation in the loss of Ca2+ transport activity with age. More generally, the project will provide a foundation for future studies designed to test strategies for the prevention and/or treatment of age-based loss of cardiac Ca2+ transport function due to oxidative damage to the Ca2+ pump. [unreadable] [unreadable] [unreadable] [unreadable]